30 Role of Long Polar Fimbriae in the Interactions of Enterohemorrhagic Escherichia coli With Peyer's Patches and Inhibition by a Probiotic Yeast

Abstract

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens associated with diarrhea, hemorrhagic colitis and life-threatening complications such as hemolytic-uremic syndrome. EHEC interact with the Follicule-Associated Epithelium (FAE) of Peyer's patches (PPs) of the distal ileum in humans and translocate across the intestinal epithelium via M cells. Molecular mechanisms are still unknown but Long Polar Fimbriae (LPF), which contribute to intestinal colonization, may be involved. Currently no specific treatment is available in EHEC infections and use of antibiotics remains controversial. Probiotic could be an alternative strategy. The objectives of the study were to investigate the role of LPF in EHEC tropism to PPs, and to explore the influence of a probiotic yeast, Saccharomyces cerevisiae, on EHEC interactions with intestinal mucosa. The expression of lpf genes (encoded by two lpf operons) of EHEC O157:H7 strain EDL933 was analyzed using in vitro models of the human upper GI tract and large intestine. To investigate the involvement of LPF in the ability of EDL933 to target PPs, we generated the D lpfA1,DlpfA2, DlpfA1-DlpfA2 isogenic mutants and trans-complemented them with lpf genes. LPF interaction with M-like cells was investigated using an in vitro model of specialized M cells. In vivo interactions of EHEC with murine PPs were analyzed in ileal loop assays. Mice were infected with a mixture of two bacterial strains, and the numbers of PPs-interacting bacteria were determined using a competitive index analysis. To investigate the effect of S. cerevisiae, mice were given the probiotic for 7 days before ileal loops assays were conducted with O157:H7 wild type. Lpf isogenic mutants (i) were not able to interact with ileal biopsies containing PPs compared to the wild type strain in competitive colonization assays and (ii) translocated across M cells at levels significantly lower than those observed for the wild type strain. Trans-complementation of the mutants with the cloned lpf genes restored their ability to interact with PPs and M cells, indicating that expression of lpfA1 or/and lpfA2 genes is required for interactions with PPs. Regarding probiotic strategy, S. cerevisiae exerts a trophic effect on the intestinal mucosa. Bloodshot PPs were macroscopically observed following EHEC infection of murine ileal loops. We showed that pre-treatment with yeast significantly inhibited O157:H7 interactions with PPs and reduced the number of hemorrhagic PPs in murine ileal loops. Yeast cell surface are rich in mannose and the role of such carbohydrates in inhibiting EHEC interactions with PPs will be investigated. We conclude that LPF are involved in the interactions of EHEC with murine PPs and are needed for an active translocation across M cell monolayer. Tropism of EHEC to PPs can be limited by a probiotic S. cerevisiae strain.